Hand-held device with reagents and method for detection and diagnostics

ABSTRACT

A hand-held device and method of processing a biological threat agent sample such that any infectious organism is rendered harmless while preserving it for subsequent testing, the method comprising placing a sample comprising a biological threat agent in a reservoir; adding a first reagent comprising peracetic acid in sufficient concentration to reach a predetermined minimal concentration after mixing with the sample in the reservoir; inactivating the sample upon interaction of the sample with the first reagent for a predetermined period of time at a predetermined temperature; removing the inactivated sample from the reservoir; and providing the inactivated sample for subsequent diagnostic testing, wherein the subsequent diagnostic testing is unaffected by inactivation of the sample. In another embodiment, the first reagent comprises a cupric salt, which is mixed with ascorbic acid and hydrogen peroxide to generate cupric ascorbate.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/494,118 filed on Jun. 7, 2011, the contents of which,in its entirety, is herein incorporated by reference.

GOVERNMENT INTEREST

The embodiments herein may be manufactured, used, and/or licensed by orfor the United States Government without the payment of royaltiesthereon.

BACKGROUND

1. Technical Field

The embodiments herein generally relate to diagnostic devices andmethods, and, more particularly, to a hand-held device with reagents andmethods for rendering safe-to-handle samples with infectious agentswhile preserving bacterial and viral signatures for immune andgenetic-based detection diagnostics and forensics.

2. Description of the Related Art

The high risk associated with biological threat agents determines thatany suspicious sample be handled under strict surety and safety controlsand processed under high level containment in specialized laboratories.These specialized facilities are complex, very expensive to operate, andneed to be staffed by personnel from an extremely limited pool ofexperts. In addition, safe means of transporting samples suspected ofcontaining highly virulent agents to specialized high level containmentlaboratories for analysis is also expensive, requiring in many countriesthe custody of armed personnel. It can be estimated that several milliondollars are spent annually worldwide to secure and safely transport anever-increasing stream of suspicious biological samples which arecollected in theatres of war, as well as in domestic environments.

As an example, U.S. Pat. No. 7,851,207 issued on Dec. 14, 2010, thecomplete disclosure of which, in its entirety, is herein incorporated byreference describes a device used to identify a variety of microbialagents simultaneously. The '207 patent is advantageous for the purposesfor which it was developed. The sample agents that are tested aregenerally not preserved for subsequent detection, diagnostics, orforensics. Moreover, the '207 patent provides denaturation andpurification steps that prevent immune-based testing. However, while the'207 patent analyses the sample using a single and specific nucleic-acidbased methodology (hybridization), it does not preserve the sample forfuture testing. Accordingly, there remains a need for diagnostictechniques that reserve potentially dangerous samples for future immuneor various nucleic acid based testing.

SUMMARY

In view of the foregoing, an embodiment herein provides a method ofprocessing a biological threat agent sample such that any infectiousorganism is rendered harmless while preserving it for subsequenttesting, the method comprising placing a sample comprising a biologicalthreat agent in a reservoir; adding a first reagent comprising peraceticacid in sufficient concentration to reach a predetermined minimalconcentration after mixing with the sample in the reservoir;inactivating the sample upon interaction of the sample with the firstreagent for a predetermined period of time at a predeterminedtemperature; removing the inactivated sample from the reservoir; andproviding the inactivated sample for subsequent diagnostic testing,wherein the subsequent diagnostic testing is unaffected by inactivationof the sample. Peracetic acid in the reservoir should be in sufficientconcentration so as to reach, after mixing with the sample, a minimalconcentration of 0.03% v/v. For example a tested effective mixtureincludes placing peracetic acid of 0.06% v/v in the reservoir to whichan equal volume of the sample is added, resulting in an active finalconcentration of 0.03% v/v. The sample and the first reagent may beplaced in the reservoir for approximately 30 minutes at approximately21° C. The method may further comprise adding a second reagentcomprising any of diluents and catalase in the reservoir to decomposeany remaining peracetic acid after inactivation. The subsequentdiagnostic testing may comprise any of a polymerase chain reaction testand an enzyme linked immunoassays test. The peracetic acid preferablycomprises 0.03% peracetic acid as a final active concentration afterdiluting with the sample.

Another embodiment provides a method of processing biological threatagents, the method comprising placing a sample comprising a biologicalthreat agent in a reservoir comprising a first reagent comprising acupric salt such as chloride or sulfate; generating in situ cupricascorbate by adding to the reservoir a second reagent comprisingascorbic acid and an amount of hydrogen peroxide that is in sufficientquantity to assure oxygenation of the mixture; inactivating the sampleupon interaction of the sample with the cupric ascorbate mixture for apredetermined period of time at a predetermined temperature; removingthe inactivated sample from the reservoir; and providing the inactivatedsample for subsequent diagnostic testing, wherein the subsequentdiagnostic testing is unaffected by inactivation of the sample. Thesample with the first reagent and second reagent may be left in thereservoir for approximately 30 minutes at approximately 21° C. Thesubsequent diagnostic testing may comprise any of a polymerase chainreaction test, nucleic acid hybridization or other nucleic acid-basedtests and an enzyme linked immunoassay, immunoprecipitation, and anyother immuno-based test. Moreover, the cupric salt may comprise cupricchloride. The first reagent may comprise a cupric salt in sufficientconcentration so as to result in a final concentration of 0.5% w/v incupric ions after dilution with the sample and the second reagent. Theconcentration of ascorbic acid should provide a final concentration suchthat mixing the sample and first reagent results in 0.1% w/v ascorbateand a small amount of hydrogen peroxide to assure aerobiosis (presenceof oxygen) in a final concentration after mixing with all others of0.003% v/vt. For example, a tested mixture resulting in concentrationswith adequate potency comprising 5 volumes of cupric chloride 2% w/v incupric ions in a first reservoir of the device, and in a secondreservoir, 4 volumes of ascorbic acid 0.5% w/v with 1 volume of hydrogenperoxide 0.06% v/v. At the time of use, 10 volumes of sample are addedto the device and all components are mixed together. The mixtureindicated above results in final concentrations of 0.5% w/v cupric ions,0.1% w/v ascorbate, and 0.003% v/v peroxide. The method may furthercomprise adding ethylenediaminetetraacetic acid to the inactivatedsample. Peracetic acid is the only biocidal reagent while cupricascorbate inactivation employs two reagents mixed in situ.

Another embodiment provides a hand-held device for sample preparation ofsamples suspected of containing biological threat agents, the devicecomprising a reservoir that holds a sample comprising a biologicalthreat agent; a first dispensing unit operatively connected to thereservoir, wherein the first dispensing unit adds a first reagentcomprising peracetic acid in sufficient concentration to reach apredetermined minimal concentration after mixing with the sample in thereservoir, wherein the sample becomes inactivated upon interaction ofthe sample with the first reagent for a predetermined period of time ata predetermined temperature; and a filter operatively connected to thereservoir and the first dispensing unit, wherein the filter removes theinactivated sample from the reservoir, wherein the inactivated sample iscapable for subsequent diagnostic testing, and wherein the subsequentdiagnostic testing is unaffected by inactivation of the sample. Thesample and the first reagent may be placed in the reservoir forapproximately 30 minutes at approximately 21° C. The device may furthercomprise a second dispensing unit operatively connected to thereservoir, wherein the second dispensing unit adds a second reagentcomprising any of diluents, catalase and a mixture of ascorbic acid andhydrogen peroxide into the reservoir. In the peracetic acid basedmethod, the peracetic acid preferably comprises 0.03% peracetic acid. Inthe cupric ascorbate based method, the cupric salt may comprise cupricchloride as the first reagent, and the second reagent may compriseascorbic acid and a small amount of hydrogen peroxide to provide, aftermixing with the sample, the final concentrations described above.

These and other aspects of the embodiments herein will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following descriptions, while indicatingpreferred embodiments and numerous specific details thereof, are givenby way of illustration and not of limitation. Many changes andmodifications may be made within the scope of the embodiments hereinwithout departing from the spirit thereof, and the embodiments hereininclude all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments herein will be better understood from the followingdetailed description with reference to the drawings, in which:

FIG. 1 is a table illustrating screening experiments with disinfectingand inactivating chemicals according to an embodiment herein;

FIGS. 2A through 2D are graphical representations illustrating thedeleterious effect of selected germicidal agents on PCR assays accordingto an embodiment herein;

FIGS. 3A and 3B are graphical representations illustrating the effect ofperacetic acid on PCR and ELISA according to an embodiment herein;

FIGS. 4A and 4B are graphical representations illustrating the effect ofcupric ascorbate on PCR and ELISA according to an embodiment herein;

FIGS. 5A and 5B illustrate a hand-held device according to an embodimentherein; and

FIGS. 6A and 6B are flow diagrams illustrating methods according to theembodiments herein.

DETAILED DESCRIPTION

The embodiments herein and the various features and advantageous detailsthereof are explained more fully with reference to the non-limitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well-knowncomponents and processing techniques are omitted so as to notunnecessarily obscure the embodiments herein. The examples used hereinare intended merely to facilitate an understanding of ways in which theembodiments herein may be practiced and to further enable those of skillin the art to practice the embodiments herein. Accordingly, the examplesshould not be construed as limiting the scope of the embodiments herein.

The embodiments herein provide a cost-effective method for specificmicrobicidal chemistry and associated hardware including a hand-helddevice to rapidly inactivate high-threat biological agents in suspectedsamples, without hindering subsequent identification. Accordingly, theembodiments herein allow for a significant cost savings of the severalmillion dollars currently spent annually to secure and safely transportan ever-increasing stream of suspicious samples suspected to containvirulent agents which are collected in military and non-militarysettings both domestically and internationally. Referring now to thedrawings, and more particularly to FIGS. 1 through 6B, where similarreference characters denote corresponding features consistentlythroughout the figures, there are shown preferred embodiments.

The embodiments herein provide unique chemical compositions that cancompletely kill all organisms being tested while preserving theperformance of detection and diagnostic methods. Experimental testing isperformed on the effect of a wide variety of microbicidal agents on: (a)the survival of vegetative bacteria, bacterial spores, DNA viruses andRNA viruses; (b) the performance of DNA-based detection and diagnosticmethods; and (c) the performance of immune-based detection anddiagnostic methods in order to develop the aforementioned chemicalcompositions.

Considerable knowledge has been accumulated regarding the efficiency ofmicrobicidal reagents and methods, particularly on inactivatingbacterial spores in liquids, on surfaces, or in aerosols but thesubsequent deleterious effect of these disinfectants on the performanceof either nucleic-acid or immune-based diagnostics precluded their usefor processing of suspected field samples. As a result, the embodimentsherein include experimental testing of two aldehydes (formaldehyde andglutaraldehyde), a halogenating agent (hypochlorite), two peroxides(hydrogen peroxide and peracetic acid), and a free radical damagingagent (cupric ascorbate). In addition, the experimental testing includeda chaotropic agent, guanidium thiocyanate, with the well-establishedability to dissociate biological structures and liberate intact nucleicacids for analysis. Spores of Bacillus (B.) atrophaeus are selected asan example microbial target because these have been frequently used insporicidal studies and also because spores of B. atrophaeus show similarsensitivity, to chemical germicides than virulent strains of B.anthracis. Cells of Pseudomonas (P.) aeruginosa are selected for testingbecause this bacterium is in the group with the highest resistance todisinfection among the vegetative cells of bacteria frequently causinghuman infection in hospitals.

To assess the effect of disinfectants on viruses, mainly on thepotential hindrance on virus detection, the Vaccinia virus (VACV) isincluded in the experimental testing, which is an orthopoxvirus (withDNA genome) generally used as a surrogate for the Smallpox virus, andPixuna virus (PIXV), which is an alphavirus that has been used as asimulant or surrogate for the Venezuelan equine encephalitis virus andother highly pathogenic RNA viruses. The inactivation of all thesemicroorganisms is followed for more than 6 log₁₀, since this is thestandard assurance level generally accepted for safety of medicaldevices and contaminated environments (based on the American ASTMStandard E-2414-05). The effect of germicidal agents is studied on theperformance of polymerase chain reaction (PCR) tests as it is one of themost frequently used nucleic acid-based diagnostic methods and an enzymelinked immunoassays (ELISA), which is one of the most widely usedimmunodetection methods.

The embodiments herein provide for the screening of chemical methodsthat completely inactivate pathogens with minimal impairment ofdiagnostics. The concentrations generally employed for chemicaldisinfection and sterilization of microbial pathogens correspond tohypochlorite 0.05% v/v (volume/volume; volume concentration),glutaraldehyde 2% v/v, peracetic acid 0.03% v/v, formaldehyde 8% v/v,hydrogen peroxide 10%, and cupric ascorbate at 0.5% in cupric ions w/v(mass/volume; mass concentration). Bacterial spores and viruses areexposed to the different chemical germicides at the followingconcentrations: hypochlorite 0.05% v/v, glutaraldehyde 2% v/v, peraceticacid 0.03% v/v, formaldehyde 8% v/v, hydrogen peroxide 10%, and cupricascorbate at 0.5% in cupric ions w/v, as well as to one lower (generallyone-tenth) and one higher (generally 10-fold) concentration.

In addition to commonly used germicides, the effect of guanidiumthiocyanate, which is known to disrupt cells and liberate nucleic acidswithout damaging them is also included in the experimental testing. Onerequirement for selection of disinfecting agents is the complete, fast,and reliable inactivation of spores at or near room temperature sinceany method to be developed should be rapid and fieldable. The results ofthese exploratory experiments with common disinfecting and inactivatingchemicals are summarized in FIG. 1. The efficiency of the reagents israted not only for their reduction of infectivity, but also for theirpreservation of immunological reactivity and nucleic acid detection.

As demonstrated by the data in FIG. 1, some chemical germicides such ashydrogen peroxide, formaldehyde, and glutaraldehyde did not completelykill all of the spores challenged in testing. These same chemicals attheir concentration of use also impair the performance of PCR and ELISAtests to detect spores and vinises as illustrated in FIG. 1.

This impairing effect on PCR and ELISA is more pronounced on virusesthan on spores (for example, see FIGS. 2A through 2D). FIGS. 2A through2D are graphical representations illustrating the deleterious effect ofselected germicidal agents on PCR assays according to an embodimentherein. More particularly, FIGS. 2A through 2D depict the deleteriouseffects of different concentrations of formaldehyde and sodiumhypochloride treatment at a reaction time of 30 min at 21° C. on PIXVand B. atrophaeus spores PCR analysis in comparison to untreatedcontrols. Microbes are treated with either water as a non-microbicidalcontrol (To) or with Formaldehyde (FA) or sodium hypochlorite (NaClO) atthe concentrations indicated inside the legend boxes in FIGS. 2A through2D. The PCR proceeded as described further below in the listing ofmaterials and methods and the relative fluorescence as a function ofnumber of amplification cycles is shown for PIXV (in FIGS. 2A and 2B) orfor spores of B. athrophaeus (in FIGS. 2C and 2D).

Formaldehyde 8% decreases the relative fluorescence of PCR testing ofspores but has only a minimal effect on their Ct-value (see FIG. 2D). Incontrast, sodium hypochlorite at the concentration generally used inliquid sterilization (0.05% v/v) completely inhibits PCR assays ofspores and viruses (see FIGS. 2B and 2C) as well as the ELISA of PIXV.Guanidium thiocyanate at 3.5 M, close to its maximal aqueous solubility,does not hinder PCR or ELISA but has little inactivating effect onspores and PIXV (see FIG. 1). Incubation at room temperature (21° C.)with either 0.5% cupric ascorbate or with 0.03% peracetic acid reducesspores and virus titer by at least 6 log₁₀ hindering diagnosticperformance of PCR and ELISA slightly except that for PIXV thepreliminary ELISA results are negative.

The embodiments herein provide for selected chemical inactivationmethods suitable for subsequent diagnostics. The results from the agentscreening shown in FIG. 1 clearly indicates peracetic acid and cupricascorbate as the two most promising chemical inactivating reagents fordevelopment of a fieldable inactivating method that will not impairsubsequent diagnostics. For further and more detailed studies VACV isalso included as a surrogate for the Smallpox virus and vegetative cellsfrom P. aeruginosa as a bacterium with relatively high resistance todisinfection. Peracetic acid, which at 0.03% v/v kills beyond detectionlevels spores >6 log₁₀ and PIXV (>7 log₁₀), affects the correspondingPCR and ELISA assays differently as shown in FIGS. 3A and 3B.

FIGS. 3A and 3B are graphical representations illustrating the effect ofperacetic acid on PCR and ELISA according to the embodiments herein. Allmicroorganisms are treated with PAA at concentrations of 0.003%, 0.03%,and 0.3%. The graph in FIG. 3A shows the effect of PAA treatment on theCt values of PCR analysis of B. atrophaeus spores (full circles), P.aeruginosa (open circles), PIXV (full squares), and VACV (open squares)relative to untreated controls. The Ct value of untreated controls are27.5±0.58 SD for B. atrophaeus, 15.8±1.16 SD for P. aeruginosa, 24.7±2.0SD for PIXV, and 23.6±2.7 SD for VACV. FIG. 3B depicts the effect of thesame treatment with peracetic acid on the corresponding ELISA. In FIGS.3A and 3B, the range of inactivation of >6 log 10 is indicated as ashaded area.

For both spores and viruses, PCR sensitivity is only marginally affected(see FIG. 3A), and the immunoassay of viruses are completely inhibitedas the inactivating concentrations (see FIG. 3B). The ELISA signalresults obtained for spores (see FIG. 3B) are only affected by PAA at aconcentration ten times higher than the concentration providing highmicrobicidal efficacy (0.03%, FIGS. 2A through 2D) and are generallyused.

FIGS. 4A and 4B are graphical representations illustrating the effect ofcupric ascorbate on PCR and ELISA according to the embodiments herein.All microbes are treated with three CuAsc concentrations of 0.1%, 0.5%,and 2.5% (in cupric ions). The range of inactivation of >6 log 10 isindicated in FIGS. 4A and 4B. The graph in FIG. 4A shows the effect oftreatment with different levels of CuAsc on PCR and RT-PCR. FIG. 4A alsoindicates the Ct values obtained by PCR analysis of B. atrophaeus spores(full circles), P. aeruginosa (open circles), PIXV (full squares), andVACV (open squares). The Ct values obtained for the untreated controlsare 28.5±0.58 SD for spores, 16.5±0.58 SD for P. aeruginosa, 24.9±0.9 SDfor PIXV, and 25.6±0.4 SD for VACV. FIG. 4B further depicts the effectof cupric ascorbate on the same viruses without chelation or afterchelation of Cu++ with 100 mM ethylenediaminetetraacetic acid (EDTA) onELISA. In FIGS. 4A and 4B, the range of inactivation of >6 log 10 isindicated as a shaded area.

Accordingly, the effect of cupric ascorbate at a concentration of 0.5%in Cu++ ions on PCR and ELISA assays is presented in FIGS. 4A and 4B,showing that the impact of disinfection on PCR assay of spores orviruses is neglectable. At a five-fold higher concentration thangenerally recommended for liquid sterilization, the PCR signal is lostfor PIXV and VACV but does not impair PCR of spores or P. aeruginosa.

The data presented in FIGS. 3A through 4B indicate that peracetic acidand cupric ascorbate increase the number of PCR cycles (relative to thenumber of cycles in the controls not exposed to germicidal agent) todetect PIXV and VACV by about 4 cycles and 0 cycles, respectively. FIGS.3A through 4B also demonstrate that no change is observed in the numberof PCR cycles to detect spores or vegetative bacteria (between 0 and 1cycle respectively) after inactivation with either peracetic acid orcupric ascorbate.

The limit of detection of ELISA tests to determine spores is marginallyreduced after treatment with peracetic acid or with cupric ascorbate. Incontrast, using ELISA to detect viruses is sensitive to disinfection(see FIG. 3B). Several substances are included in the experiments in anattempt to minimize the impairing effect of peracetic acid and cupricascorbate on viral ELISA. The addition to post disinfection mixes ofcatalase (32 to 320 units) or of Tris-EDTA, pH8, in order to degrade theremaining peroxide radicals in peracetic acid or neutralize acid cannotrestore the PIXV ELISA signal after disinfection with peracetic acid.Among all the substances under testing, EDTA best prevents theimpairment of viral ELISAs by disinfection with cupric ascorbate. Theresults shown in FIG. 4B indicates that cupric ascorbate withoutpost-treatment with EDTA completely inhibits immunological reactionswith the PIXV and the VACV. However, the addition of EDTA, pH8, up to afinal concentration of 100 mM after the disinfection with cupricascorbate protects the ELISA signal from VACV (a virus whose genome isDNA) to about 90% compared to the untreated sample. Immunoassays todetect PIXV (a virus with RNA genome) are more sensitive to cupricascorbate since EDTA retains only about 20% of the signal compared tothe untreated antigen. These results suggest that viral PIXV antigensare more sensitive to oxidation and inactivation by cupric ascorbatethan VACV antigens.

Peracetic acid reduces the sensitivity of spores immunoassays to nearly20% of the results obtained with untreated controls, while cupricascorbate has no impact on these assays. Moreover, inactivation ofvegetative bacteria, bacterial spores, or viruses with these reagentsdoes not lead to false-positive signals in subsequent PCR or ELISAtesting.

As shown in FIGS. 5A and 5B, with respect to FIGS. 1 through 4B, theembodiments herein further include a hand-held device 50 to inactivatemicrobes in suspected samples without hindering subsequent diagnosticsor detection. The device 50 is dimensioned and configured to accommodateone or more of the reagents described above. The device 50 includes asample reservoir 52 (for example, a jar or bottle with or without a rib57, etc.) where the sample is resuspended or diluted in saline, and ahead unit 54 which operatively connects to two dispensing mechanisms 56,58 (for example, syringes) for dispensing reagents. The head unit 54further includes a filter 60, gripping ribs 62, a pair of conduits 64,66, and seal 68. The reservoir 52 may include threads 53 to engage withcorresponding threads (not shown) of the head unit 54 to create aconnection between the reservoir 52 and head unit 54. A correspondingseal 55 may be included on the reservoir 52, which may align with theseal 68 of the head unit 54 to create a liquid-tight connection betweenthe reservoir 52 and head unit 54. The gripping ribs 62 allow a user torotate the head unit 54 to open/close the device 50.

In a first embodiment, using peracetic acid as described above, one ofthe dispensing mechanisms 56 (for example) contains peracetic acid andthe other dispensing mechanism 58 (for example) can contain diluents,catalase, or left empty. The sample is introduced in the reservoir 52and mixed with saline, for example. Dispensing mechanism 56 withperacetic acid is emptied into reservoir 52 via conduit 64 and is leftfor approximately 30 minutes at room temperature. Afterwards,discharging dispensing mechanism 58 (via conduit 66) with catalase isoptional depending on the type of subsequent analysis/diagnostics to beperformed. The device 50 is then inverted and the inactivated sample isaspirated through the filter 60 and into dispensing mechanism 58 (viaconduit 66). Dispensing mechanism 58 with the inactivated and filteredorganisms is removed from the device 50 and subjected to furtheranalysis/diagnostics under low containment requirements.

In a second embodiment, cupric chloride or other cupric salt is indispensing mechanism 56 and a proper mixture of ascorbic acid and lowamounts of hydrogen peroxide are in dispensing mechanism 58. Operationof the device 50 is the same as described in the first embodiment withthe subsequent addition of the treated samples to EDTA used as apreservative.

Seven commonly used liquid disinfectants are experimentally tested inaccordance with the embodiments herein. The majority of these substanceseither partially inactivates the microbial load or severely impairssubsequent PCR and ELISA tests. The embodiments herein provide a rapid,reliable, and straightforward method for the complete inactivation of awide range of pathogens including spores, vegetative bacteria, andviruses, while preserving microbial nucleic acid fragments suitable forPCR reactions and proteinaceous epitopes for the detection ofimmunoassays. The experimental data demonstrates that a high levelinactivation (more than 6 log₁₀) of vegetative bacteria, bacterialspores, and DNA or RNA-viruses can be attained within approximately 30minutes at 21° C. with either peracetic acid (0.03%) or cupric ascorbate(0.5% in Cu++) treatment with only minimal hindrance in the subsequentperformance of PCR.

Although sensitivity of immunoassays depends on the affinity andconcentration of available antibodies, the numerous ELISA that areexperimentally performed with diverse liquid disinfectants and variousmicrobes provide support to the conclusion that, in general, PCR assayswithstand treatment with a variety of disinfectants better thanimmunoassays.

Peracetic acid disinfection maintains ELISA sensitivity from 84% to 90%of that of untreated controls during the detection of spores andvegetative bacteria, nearly 50% for detection of VACV, and completelyinhibited ELISA detection of PIXV.

Disinfection with cupric ascorbate preserves the sensitivity of ELISAfor vegetative bacteria and bacterial spores (within 80-90% of untreatedcontrols) but without further treatment impairs the results of viralimmunoassays. The addition of EDTA after incubation with cupricascorbate preserves the signal in ELISA to detect VACV at nearly 90% ofthat in untreated controls and at 20% of the signal obtained with PIXV.Inactivation does not lead to false-positive signals. These signallevels are adequate for environmental detection and identification ofenvironmental samples.

The relatively rapid inactivation of the high microbial loads in theexperimental samples at room temperature by cupric ascorbate orperacetic acid used as described herein are effective means of quicklyrendering field samples suspected of containing infectious agents safefor further analysis under lower containment, and at considerably lowercosts. Moreover, sample disinfection with peracetic acid is simple andcan be used before shipping suspected samples to those laboratoriesrelying exclusively on PCR methods for the rapid detection of hazardousinfectious agents. However, decontamination with peracetic acid shouldbe selected only if certain that immunoassays will never be performed onthe disinfected samples since the inhibition of immunoassays byperacetic acid is considerable.

Although an additional step involving the addition of EDTA is used topreserve immunodiagnostic performance, the relatively rapid and completeinactivation of all tested microbes at room temperature by cupricascorbate appears as the most promising method to render field samplesnon-infectious and thus easily and safely transportable for subsequentanalysis and diagnostics by PCR and/or immunodiagnostics. Some loss inimmune reactivity for viruses should be expected during disinfection ofsamples with either cupric ascorbate or peracetic acid, but this resultshould be more than compensated by the concomitant gains in surety,safety, and economy resulting from handling samples as non-infectiousand thus, at a much lower containment level.

The experimental materials and methods used in accordance with theembodiments herein are described below. The listed materials and methodsare examples only, and the embodiments herein are not limited to theseparticular materials and methods.

Microbial species and sources: Bacillus atrophaeus (strain ATCC 9372) isobtained from the American Type Culture Collection (USA). Spores of B.atrophaeus are prepared in accordance with the DIN EN 14347 (EN 14347,2005. Chemical disinfectants and antiseptics. Basic sporicidal activity.Test method and requirements; phase 1, step 1) and resuspended at aconcentration of 2×10⁸ spores per mL. P. aeruginosa (strain DSm 1253) isobtained from the German strain culture collection (Deutsche Sammlungvon Mikroorganismen and Zellkulturen GmbH [DSMZ], Braunschweig,Germany). On the day previous to each experiment, one colony of P.aeruginosa originally isolated in Tryptone-Soya-Broth (TSA, Oxoid,Wesel) is seeded into 50 ml media and grown for approximately 20 hoursat 37° C. with agitation. On the day of the experiment, cells near theend of their exponential phase of growth are washed three times bycentrifugation and resuspended in sterile distilled water. The titer ofP. aeruginosa in the final working suspension ranges from 0.8 to 2.4×10⁹cfu/mL. PIXV strain BeAr 35645; Brazil 1961, ATCC # VR-371, ispropagated in Baby Hamster Kidney cells (BHK cells, DSKZ-ACC 33, DMSZ,Braunschweig, Germany) at 37° C. in Eagle's Minimum Essential Medium(EMEM) available from Biochrom (Berlin, Germany), containing 10% FCS and0.1% penicillin/streptomycin and harvested from infected cell monolayersat a concentration of 1.2×10¹⁰ TCID₅₀/ml. VACV strain Elstree B5/Lister(WHO reference strain; Czerny and Mahnel, 1990) is grown in BHK cells at37° C. in Eagle's Minimum Essential Medium (EMEM) containing 10% FCS and0.1% penicillin/streptomycin and used at a TCID₅₀/mL ranging from 2×10⁶to 4×10⁶.

Chemical disinfectants: Formaldehyde and guanidium thiocyanate areavailable from Roth GmbH (Karlsruhe, Germany), with the later beingdissolved at a concentration of 7 M in sterile distilled water and itspH adjusted between 7 to 8 at room temperature with Tris-HCl. Peraceticacid and sodium hypochlorite containing 6.7% active chlorine asdetermined by iodometric determination are available from Sigma-Aldrich(Taufkirchen, Germany): Before use, the hypochlorite solution isadjusted to pH 7.5 with hydrochloric acid. Glutaraldehyde available fromSigma-Aldrich (Taufkirchen, Germany) is activated before each experimentby adjusting it to pH 9.3 with sodium bicarbonate. Hydrogen peroxide 30%is available from Merck—VWR International, Darmstadt, Germany. Cupricchloride dehydrate (>99% ACS reagent (Sigma-Aldrich, Taufkirchen,Germany)) and 0.003% hydrogen peroxide; with this last substance beingadded to assure instant liquid oxygenation for efficient free radicalgeneration.

Microbial inactivation: A microbial suspension (typically 50 μl)containing either 1×10⁸ spores×mL⁻¹, 0.8 to 2.4×10⁹ P. aeruginosacells×mL⁻¹, 1.2×10¹⁰ PIXV Tissue Culture Infectious Dose 50(TCID₅₀)×mL⁻¹, or 4×10⁶ VACV TCID₅₀×mL⁻¹ is dispensed into 1.5 mlEppendorf type tubes carefully avoiding microbial contamination of theinside walls of the tube above liquid level. An equal volume of eitherPBS as a control without inactivating effect or disinfectant at variousconcentrations is added and the mixture with microbes and disinfectantis incubated at 21° C. for 30 minutes with a 10 second initial mixing at300 rpm (Eppendorf Thermomixer comfort, Eppendorf AG., Hamburg,Germany). Only cupric ascorbate is prepared in situ by adding to 50 μlof microbial suspension, 25 μl cupric chloride at 4-fold the intendedfinal concentration, followed by 20 μl of ascorbic acid at 5×, andfinally 5 μl of hydrogen peroxide at 20× the intended finalconcentration. After disinfectant treatment, nine volumes (900 μl) ofeither ice-cold Tryptone Soya Broth (TSB, Oxoid, Wesel, Germany) forbacteria or ice-cold EMEM with 10% fetal calf serum for viruses areadded to the treatment mixes in order to slow down any remaininginactivation process. Surviving bacteria are analyzed in aliquots byserial dilution and titration onto TS agar plates. Viruses are seriallydiluted in EMEM supplemented with 5% fetal calf serum and in dilutionsranging from between 10⁻⁵ to 10⁻⁸ plated on to BHK cell monolayers in96-well cell culture microtest plates. The medium is removed from cells,and the virus in the sample (25 μl) is adsorbed for one hour. Theinoculum is removed and replaced with fresh EMEM with 10% FCS (25 μl perwell) before the plates are incubated overnight at 37° C. in 4% CO₂atmosphere. The amount of surviving virus is determined by the TCID₅₀method on BHK cell monolayers. Other aliquots of the same exposed sampleare tested by PCR and ELISA as described below.

DNA and RNA preparation: Nucleic acids, DNA, or RNA from VACV or PIXV,respectively, are extracted from a 100 μl aliquot of the sampleinactivated with the studied disinfectants. Purification is performed byusing either the QIAamp® DNA Mini Kit or the QIAamp® Viral RNA Mini Kit(Qiagen, Hilden, Germany) according to the manufacturer's instructionswith minor modifications. Viral DNA and RNA are eluted with 100 μl ofthe cognate buffer. Generally, 1 μl of DNA is subjected to the VACVspecific real-time PCR (rtPCR) and 5 μl of the cognate buffer.Generally, 1 μl of DNA is subjected to the VACV specific real-time PCR(rtPCR) and 5 μl RNA is subjected to the PIXV specific real-time ReverseTranscriptase PCR (rtRT-PCR).

rtPCR and rtRT-PCR: For real-time PCR either the one-step RT-PCR, theHotStarTaq® system, or the TaqDNA Polymerase system from Qiagen GmbH oreither ice-cold Tryptone Soya Broth (TSB, Oxoid, Wesel, Germany) forbacteria or (Hilden, Germany) chemistries are used with either TaqMan-or SYBR Green1 (TIB Molbiol GmbH, Berlin, Germany), respectively. Thefluorescent reporter dye of the viral probe is a 6-carboxyfluorescent(FAM) located at the 5′ end in all 5′-nuclease assays. The quencher, a6-carboxy-tetramethyl-rhodamine (TAMRA), is located at the 3′ end.Purified nucleic acids are used in viral PCR assays and extensivelywashed and resuspended spores or vegetative cell suspensions aredirectly applied to the respective PCR test. The rtPCR as well as thertRT-PCR assays are performed in microtest plates (Biozym ScientificGmbH, Germany) in a final volume of either 25 μl or 20 μl on an Opticon™device Type 1 (BioRad, Laboratories Inc., USA). Primer-, 5′-nucleaseprobe, SYBR Green I, Mg⁺⁺- and dNTP-concentrations are optimized bytitration. Each real-time PCR and RT-PCR is performed in duplicates ortriplicates and individual experiments are repeated two to four times.Negative controls contain water instead of a potential nucleic acidtemplate. Impairment of disinfectants on either PCR or RT PCR tests arecorrelated to the C_(t) (Cycle threshold), which corresponds to thefirst cycle number in which the fluorescence signal significantlyincreases from the baseline and background.

Enzyme-linked immunoassay (ELISA): All ELISAs are performed in 96microwell plates (Maxisorb™, Thermo Fisher Scientific, Dreieich,Germany) that are coated with 3 to 4 μg antibody per well either byincubation over night at 4° C. or by incubation for two hours at 37° C.while plates for viral ELISAs are washed and blocked with 1% FCS inPBS-T (PBS plus 0.01% Tween 20) for 1 hour at room temperature (RT).Plates for bacterial ELISAs are blocked with 1% low-fat-milk powder inPBS for 30 minutes at RT. In addition, plates for viral ELISA areoverlayed with liquid plate sealer (Candor Bioscience GmbH, Wangen,Germany) and are used for ELISA studies within three to four weeks.Plates for bacterial ELISAs are used and freshly coated each time.

For the detection of PIXV, the species specific monoclonal antibodyPixcT 6/2 are used (available from the University of VeterinaryMedicine, Hannover, Germany) as the capture antibody. For the detectionof VACV, an equimolar mixture of the monoclonal antibodies mAB 5B1 and5B4 can be used. P. aeruginosa and B. atrophaeus endospores are capturedwith specific rabbit polyclonal (pAb) antibodies produced in alaboratory setting. Antigen incubation is performed for either one ortwo hours at room temperature (21° C.) or at 37° C. Bound viral andbacterial antigens are detected by using either biotinylated speciesspecific monoclonal (mAb), WIS pAb anti-P. aeruginosa 1:400) and afterextensive washing the conjugate Streptavidin-horseradish peroxidase(PSA, available from GE Healthcare, USA) is added to the wells diluted1:6000 in PBS-FT. Plates are incubated for 30 min at 21° C. withagitation and after three washes with PBS-T, staining is performed withthe colorimetric substrate 3-3′,5,5′-tetramethylbenzidine (TMB,available from Serva, Heidelberg, Germany) for 10 minutes. Furthermore,color development is stopped with 2 M sulphuric acid and absorbance ismeasured at 450 nm. Improvement of ELISA results obtained after cupricascorbate disinfection is evaluated by the addition of EDTA, (pH8, in afinal concentration of 2, 10, 20, 40, and 100 mM). The enhancement ofELISA results after peracetic acid can be attempted by the addition ofeither 1 M Tris, pH 8, or catalase in a final concentration ranging from32 to 324 units). All three potential ELISA enhancers (EDTA, Tris, andcatalase) are available from Sigma Aldrich, Germany and are added tosamples after the 30 min inactivation with disinfectants.

FIG. 6A, with reference to FIGS. 1 through 5B, is a flow diagramillustrating a method of processing biological threat agents, wherebythe method comprises placing (161) a sample comprising a biologicalthreat agent in a reservoir 52; adding (163) a first reagent comprisingperacetic acid in sufficient concentration to reach a predeterminedminimal concentration after mixing with the sample in the reservoir 52;inactivating (165) the sample upon interaction of the sample with thefirst reagent for a predetermined period of time at a predeterminedtemperature; removing (167) the inactivated sample from the reservoir52; and providing (169) the inactivated sample for subsequent diagnostictesting, wherein the subsequent diagnostic testing is unaffected byinactivation of the sample. The peracetic acid in the reservoir 52should be in sufficient concentration so as to reach, after mixing withthe sample, a minimal concentration of 0.03% v/v. For example a testedeffective mixture includes placing peracetic acid of 0.06% v/v in thereservoir 52 to which an equal volume of the sample is added, resultingin an active final concentration of 0.03% v/v. The sample and the firstreagent may be placed in the reservoir 52 for approximately 30 minutesat approximately 21° C. The method may further comprise adding a secondreagent comprising any of diluents and catalase in the reservoir 52 todecompose any remaining peracetic acid after inactivation. Thesubsequent diagnostic testing may comprise any of a polymerase chainreaction test and an enzyme linked immunoassays test. The peracetic acidpreferably comprises 0.03% peracetic acid as a final activeconcentration after diluting with the sample.

FIG. 6B, with reference to FIGS. 1 through 6A, is a flow diagramillustrating another method of processing biological threat agents,whereby the method comprises placing (261) a sample comprising abiological threat agent in a reservoir 52 comprising a first reagentcomprising a cupric salt; generating (263) in situ a cupric ascorbatemixture by adding to the reservoir 52 a second reagent comprisingascorbic acid and an amount of hydrogen peroxide that is in sufficientquantity to assure oxygenation of the mixture; inactivating (265) thesample upon interaction of the sample with the cupric ascorbate mixturefor a predetermined period of time at a predetermined temperature;removing (267) the inactivated sample from the reservoir 52; andproviding (269) the inactivated sample for subsequent diagnostictesting, wherein the subsequent diagnostic testing is unaffected byinactivation of the sample. The sample with the first reagent and thesecond reagent may be placed in the reservoir 52 for approximately 30minutes at approximately 21° C. The subsequent diagnostic testing maycomprise any of a polymerase chain reaction test, nucleic acidhybridization, other nucleic acid-based tests, an enzyme linkedimmunoassay, immunoprecipitation, and any other immuno-based tests. Thecupric salt may comprise any of cupric chloride and sulfate. The firstreagent preferably comprises the cupric salt in a sufficientconcentration so as to result in a final concentration of 0.5% w/v incupric ions after dilution with the sample and the second reagent. Themethod may further comprise adding ethylenediaminetetraacetic acid tothe inactivated sample.

The high risk associated with biologically threat agents determines thatany suspicious sample should be handled under strict surety and safetycontrols and processed under high level containment in specializedlaboratories. Accordingly, the embodiments herein provide a rapid,reliable, and simply method and device for the complete inactivation ofa wide range of pathogens including spores, vegetative bacteria, andviruses, while preserving microbial nucleic acid fragments suitable forPCR reactions and other nucleic acid-based methods and proteinaceousepitopes for the detection by ELISA or other immunoassays. Conventionalmethods and devices for providing disinfectants either do not inactivatecompletely high titers of bacterial spores or viruses, or show highmicrobicidal activity but obliterate future PCR or ELISA diagnostictesting to detect bacterial spores or viruses. Among all theformulations experimentally tested, only high level inactivation (morethan 6 log₁₀) of bacterial spores (B. atrophaeus), vegetative bacteria(P. aeruginosa), an RNA-virus (the Alphavirus PIXV), or a DNA-virus (theOrthopoxvirus VACV) is attained within 30 minutes at 21° C. by treatmentwith either peracetic acid or cupric ascorbate with minimal hindrance ofsubsequent PCR tests and immunoassays. The method and device provided bythe embodiments herein enable quick rendering of field samples ofbiological agents and permit further analysis under lower containmentand at a lower cost than conventional solutions.

Accordingly, the embodiments herein provide liquid reagents with highefficiency to inactivate microbial organisms and viruses thatsimultaneously preserve the high sensitivity of subsequent diagnosticsperformed at a lower level of containment and cost. The identifiedreagents are integrated into a self-contained device for inactivation ofmicrobes in environmental samples for subsequent detection anddiagnostics.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the embodiments herein that others can, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without departing from thegeneric concept, and, therefore, such adaptations and modificationsshould and are intended to be comprehended within the meaning and rangeof equivalents of the disclosed embodiments. It is to be understood thatthe phraseology or terminology employed herein is for the purpose ofdescription and not of limitation. Therefore, while the embodimentsherein have been described in terms of preferred embodiments, thoseskilled in the art will recognize that the embodiments herein can bepracticed with modification within the spirit and scope of the appendedclaims.

What is claimed is:
 1. A method of processing biological threat agents, said method comprising: placing a sample comprising a biological threat agent in a reservoir comprising a first reagent comprising a cupric salt; generating in situ a cupric ascorbate mixture by adding to said reservoir a second reagent comprising ascorbic acid and an amount of hydrogen peroxide that is in sufficient quantity to assure oxygenation of the mixture; inactivating said sample upon interaction of said sample with said cupric ascorbate mixture for a predetermined period of time at a predetermined temperature; removing the inactivated sample from said reservoir; and providing the inactivated sample for subsequent diagnostic testing, wherein said subsequent diagnostic testing comprises immune or genetic-based testing used to detect or identify the biological threat agent and which is unaffected by inactivation of the sample.
 2. The method of claim 1, wherein said sample with said first reagent and said second reagent are placed in said reservoir for approximately 30 minutes at approximately 21° C.
 3. The method of claim 1, wherein said subsequent diagnostic testing comprises any of a polymerase chain reaction test, nucleic acid hybridization, other nucleic acid-based tests, an enzyme linked immunoassay, immunoprecipitation, and other immune-based tests.
 4. The method of claim 1, wherein said cupric salt comprises any of cupric chloride and sulfate.
 5. The method of claim 1, wherein said first reagent comprises said cupric salt in a sufficient concentration so as to result in a final concentration of 0.5% w/v in cupric ions after dilution with said sample and said second reagent.
 6. The method of claim 1, further comprising adding ethylenediaminetetraacetic acid to the inactivated sample. 